In such fields as microbiology, histology, and pathology samples containing biological matter such as individual cells or tissue samples are routinely investigated using microscopy, in particular transmission microscopy. The material to be imaged is usually stained in order to increase the colour contrast between different types of its constituents. Without staining, it is very difficult to see differences in cell morphology. Hematoxylin and eosin (H & E) are the most commonly used stains in histology, pathology, and microbiology. Hematoxylin colours nuclei blue, eosin colours the cytoplasm pink. For observing the tissue under a microscope, the sections are stained with one or more pigments.
There are hundreds of various special staining techniques which have been used to selectively stain cells and cellular components. Compounds for colouring tissue sections include safranin, oil red o, congo red, fast green FCF, silver salts and numerous natural and artificial dyes, which usually originate from the development of dyes for the textile industry.
Recently, antibodies have been used to visualize specifically proteins, carbohydrates and lipids. This is known as immunohistochemistry, or, when the stain is a fluorescent molecule, immunofluorescence. This technique has greatly increased the ability to identify categories of cells under a microscope. It can be combined with other advanced techniques, such as non-radioactive in-situ hybridization for identifying specific DNA or RNA molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-linked fluorescence amplification. Fluorescence microscopy and confocal microscopy are used to detect fluorescent signals with good intracellular detail. Digital cameras are increasingly used for capturing histological and histopathological images.
A problem is that often biologists and pathologists are used to working with a certain staining method used at their laboratory and have trouble interpreting images resulting from different staining methods due to the fact that different staining methods result in different colours of the samples. For example, one pathologist is used to applying H & E at a certain ratio, while a second pathologist uses another ratio of H & E. The second pathologist may also routinely use a staining method other than H & E. In both cases the two pathologists may find it difficult to interpret each other's images.
T. Abe, et al. [“Colour correction of pathological images based on dye amount quantification”, Optical Review 12, 293-300 (2005)] have proposed a colour correction method for pathological images of H & E stained samples in which the amounts of H & E dyes are estimated based on a multispectral imaging technique using the Beer-Lambert law. The colour image is generated in accordance with an adjusted amount of dyes. Thereby an image can be corrected to an arbitrary or specified optimal staining condition image. The document also describes using colour management techniques for correcting pathology images for differences between computer displays and microscopes.
In digital imaging systems, colour management is the controlled conversion between the colour representations of various devices, such as image scanners, digital cameras, monitors, TV screens, film printers, computer printers, offset presses, and corresponding media. The primary goal of colour management is to obtain a good colour match across different devices. For example, a video should appear in the same colours on a computer LCD monitor, a plasma TV screen, and a printed frame of video. Colour management helps achieving the same appearance on all of these devices, provided the devices are capable of delivering the needed colour intensities.
A disadvantage of the method proposed by Abe et al is that the spectral analysis involves a considerable experimental effort and sophisticated equipment. Furthermore it is not evident how the method could be used to convert colours resulting from H & E into colours resulting from a different type of dye.
It is an object of the invention to provide a method of determining a colour transformation for images of biological material without requiring knowledge about the way in which the biological material has been prepared. It is another object of the invention to provide a data carrier carrying an image file that allows the image to be displayed alternatively in the colours resulting from a first preparation method or in the colours resulting from a second preparation method.
These objects are achieved by the features of the independent claims. Further specifications and preferred embodiments are outlined in the dependent claims.